The presence of substantial pools of cholesteryl esters in the adrenal cortex and ovary, and the recognition that this potential substrate enters the steroidogenic pathway as free cholesterol, signifies the important role of cholesterol esterase in the regulation of steroidogenesis. The neutral cholesteryl ester hydrolase (CEH) activity purified from the adrenal and the ovary appears to be identical to hormone-sensitive lipase (HSL) purified from adipose tissue. While significant information has been generated to advance the understanding of some of the mechanisms acutely regulating HSL activity in adipose tissue, very little data exist concerning changes in the expression of CEH and the factors and mechanisms regulating its expression in steroidogenic tissues. Similarly, the consequences of the action of HSL as a neutral cholesterol esterase have not been directly established in regard to influencing steroidogenesis. Moreover, the regulation of neutral CEH activity has been shown to be rapidly modulated by hormones via phosphorylation-dephosphorylation reactions, yet, very little information exists on the mechanisms involved in regulating HSL other than posttranslational modification. To address these issues, the overall goals of this proposal are to understand the mechanisms regulating the expression of neutral CEH in steroid-producing cells and to establish the structure-function relationships of HSL as a neutral cholesterol esterase. The first aim is to explore the mechanisms regulating the physiological expression of neutral CEH in steroidogenic cells. The mechanisms regulating neutral CEH in vivo and in vitro in steroidogenic cells will be examined by following changes in CEH activity, the amount of HSL protein, the steady-state mRNA levels, and the rate of transcription of HSL mRNA in the adrenal and ovary during relevant hormonal manipulations. In addition, the mechanisms responsible for activation of HSL-mediated cholesteryl ester hydrolysis by trophic hormones will be explored by examining the possible translocation of HSL. The second aim is to establish the structure-function relationships of HSL as a neutral cholesteryl esterase in steroidogenic cells and its subsequent effects on steroidogenesis. Murine adrenal cell lines and transformed rat granulosa cells will be stably transfected, while primary cultures of adrenal, granulosa, and luteal cells will be transiently transfected, to overexpress normal HSL and the ability of the cells to accumulate cholesteryl esters and to produce steroids will be examined. CHO, adrenal, and granulosa cell lines will be transfected with mutated forms of rat HSL to examine the function of HSL and the structural relationships of phosphorylation and the putative lipid binding domain. Finally, steroidogenic cell lines will be stably transfected with an antisense hammerhead ribozyme construct, or antisense oligonucleotides will be used in primary cultures of adrenal, granulosa and luteal cells, to eliminate HSL expression to examine the importance of HSL in cholesteryl ester hydrolysis and its actions on steroidogenesis. The results of this proposal will delineate the function of HSL, the mechanisms controlling its expression, and its contribution to the regulation of steroidogenesis-linked cholesterol ester hydrolysis.